Antibacterial Property of Santol (Sandoricum koetjape) Leaf

Extract Against Staphylococcus Aureus

Research Plan

A. Question or Problems being addressed

Cellular phones can be seemingly considered as an extension of our human body. People

tend to bring them everywhere they go even in bathrooms. With that being said, the frequent

exposure of these phones makes it highly susceptible for bacterial growth and transfer. A certain

research from the Bulletin Pharmaceutical Research showed that E.coli, E. aerogenes,

Streptococci and Staphylococcus aureus in the percentage of 23.53%, 23.53%, 17.65%, and

35.30% respectively were found on phones. (Verma, D.K., 2015)

But aside from smartphones, Staphylococcus spp can also be found in the human skin

particularly in the hands and on our faces which causes pimples build up. According to Otto

(2010), staphylococci are the most abundant skin-colonizing bacteria and the most important

causes of nosocomial infections and community-associated skin infections. The Staphylococcus

genus includes at least 40 species. Of these, nine have two subspecies, one has three subspecies,

and one has four subspecies.

The ingestion of these bacteria can be very detrimental to one’s health. Disinfecting these

microorganisms is needed to avoid its growth that may cause harmful diseases. The success of

this research can help the community in terms of health care and as well as every cell phone user

since it can minimize the risk of acquiring diseases caused by bacteria found on their cellular

phones and skin.

The main goal of this research is to discover the efficacy of the Santol leaf extract against

Staphylococcus aureus with the significant components found in Santol leaf extract that are

utilizable for the elimination of bacteria. The study aims to determine the effectivity of oregano

extract in dissolving these bacteria, specifically the Staphylococcus aureus, found in skins as

well as contaminated cellular phones and the researchers will also identify if the bacteria

acquired were gram positive or gram negative.

Generally, the problem that was addressed in this problem is about an individual’s health

and sanitary concerns. The ingestion of these bacteria can be very detrimental to one’s health.

Disinfecting these microorganisms is needed to avoid its growth that may cause harmful

diseases. With this research, the researchers hope to create an effective alternative and organic

bio-bactericide. The success of this research can help the RTNHS students and as well as every

cell phone user since it can minimize the risk of acquiring diseases caused by bacteria found on

their cellular phones and in the human skin.

This study specifically aims to answer the following questions:

1. Does santol leaf extract contain the key ingredients in removing Staphylococcus spp?

2. Is there a significant difference among the Staphylococcus aureus treated with various

concentration of santol leaf extract in terms of radial growth and percent of inhibition?

3. Is there a significant difference among the Staphylococcus aureus treated with commercial

bactericide and with the various concentration of santol leaf extract in terms of radial growth and

percent of inhibition?
4. Can the santol leaf extract be an effective bio-bactericide to fight against Staphylococcus

bacteria?

Hypothesis

1.The oregano leaf extract does not contain any ingredient in fighting against Staphylococcus

aureus.

2. The santol leaf extract has no significant difference among the Staphylococcus spp treated

with various concentrations of santol leaf extract and with the commercial bactericide, therefore

making it as an ineffective bio-bactericide.

C. Procedures

Materials

Chemical reagents and Treatments used:

Treatment A: 3 mL Pure Santol Leaf Extract + Nutrient Agar

Treatment B: 2.4 mL (80%) Santol Leaf Extract + 0.5 mL (20%) Water + Nutrient Agar

Treatment C: 1.8 mL (60%) Santol Leaf Extract + 1.2 mL (40%) Water + Nutrient Agar

Treatment D (Negative Control): 3 mL Distilled Water + Nutrient Agar

Treatment E (Positive Control): 10 mL Antibiotic (Commercialize) + Nutrient Agar

7 grams Nutrient Agar

Distilled Water

Methylene Blue

Ethanol

Iodine
Carbol fuchsin

Alcohol

Apparatuses and Equipments used:

Petri Dishes

1 Incubator

3 Forceps

1 Inoculating Loop

1 Alcohol Lamp

2 Water Glass

1 Pipette with Tips

1 Spreader

1 Stirring Rod

2 Gloves

2 Masks

1 Vernier Caliper

2 Glass Slides

2 Microscopes

Improvised Antibiotic Sensitivity Disc

Wire Staining Screen

Whatman Disks

C.1 Authentication of Santol Leaves

authenticated at the Bureau of Plant Industry at Bacolod.

C.2 Collection and Drying of Santol leaves

300 grams of santol leaves will be collected at the researcher's residence and it will be washed

thoroughly with distilled water. The leaves will be air dried in a closed room for 3 days.

C.3 Preperation and Extraction of Santol (Crude) Leaf Extract

275 grams of powderized santol will be macerated with 150mL of ethanol for 24-48 hours. The

pure extract will be seperated from the ethanol through a Rotatory Evaporator.

C.4 Inoculum Preparation

An inoculating loop will be used to scrape off enough bacteria sample from its colony and

will be mixed with a 20 mL distilled water. The mixture is called an inoculum. Fifteen petri

dishes were then prepared with labels of the treatments. For each treatment, there are three

replicates and were labelled namely P. Santol (Pure Santol leaf extract + Nutrient Agar); 80%

(2.4 mL (80%) Santol leaf Extract + 0.5 mL (20%) Water + Nutrient Agar); 60% (1.8 mL (60%)

Santol leaf Extract + 1.2 mL (40%) Water + Nutrient Agar); DW (3 mL Distilled Water +

Nutrient Agar); and A (10 mL Antibiotic (Commercialize) + Nutrient Agar).

C.5 Disc Diffusion Assay

The nutrient agar will be added to the petri dishes using the pipette followed by the

inoculum. After that a spreader will be sterilized under the process of flaming, then it will be

cooled and will be used to spread out the bacteria in the dish evenly. The improvised antibiotic

sensitivity discs were then soaked in the various concentration of the santol leaf extract and were
placed with each petri dishes depending on the label. 3 discs were placed in every petri dishes

with 3 cm apart. After closing the petri dishes, the lid of the covers were then sterilized to kill

excess microorganisms.

C.6 Storing of the Treatments

After the Disk Diffusion Assay procedure, the Treatments will be then stored in the oven

dryer for about 12 hours so that the bacteria can inhibit the petri dishes.

C.7 Gathering of Data According to the Zone of Inhibition

After 12 hours, the petri dishes will be then pulled out and it is ready for measuring.

Using the Vernier Caliper, the zone of inhibition of each treatments will be measured in

millimeters and the data were recorded.

C.8 Gram Staining

A smear will be prepared from the given bacterial culture and were placed in two glass

slides. Two slides will be placed on the wire staining screen and will be flooded with methylene

blue for 1 minute. After staining for 1 minute, the methylene blue will be washed with tap water

and the excess water was drained off. After that, the two glass slides with smear will be then

flooded with iodine for another minute. After one minute, these will be washed with tap water

and will be lightly blotted with tissue paper to remove excess water but it will be not completely

dried. The slides will be then tilted and were decolorized with 95%alcohol for about fifteen

seconds. The slides will be washed again with tap water. The smears were then counter-stained

with carbol fuchsin for about 30 seconds and will be washed with tap water. The slides will be
examined under the microscopes and will be identified if it will be gram positive or gram

negative. The structure and color or the bacteria Staphylococcus will be observed.

C.9 Proper Disposal

For standard sanitary measures, the researchers will see to it that the used treatments and

used experimental materials will be properly disposed.

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